Sinonasal epithelial cells (SNEC) line the sinus cavity and play important roles in orchestrating innate and adaptive immune responses. We, and others, have demonstrated that SNEC from patients with chronic rhinosinusitis (CRSwNP), are endogenously pro-inflammatory, hyper-responsive to environmental stimuli, and promote immune cell infiltration and activation. Recently, multiple components of the complement (Cp) system have been shown to be activated and up regulated in the mucosa of CRSwNP patients, including C3, though how they contribute to CRS-related inflammation is unknown and thus the focus of these proposed studies. Traditionally, the observed effect of Cp factors and receptors on various cell types was thought to be mediated solely by Cp activation fragments generated in the serum, the lymph, or interstitial fluids. Yet, recent paradigm shifting studies have shown that Cp generated by the cell itself can function in an autocrine, and unexpectedly, an intracellular fashion, and that intracellular signaling is essential for regulating the cells functions. We have shown that SNEC cells are the primary sinonasal cavity producers of C3, the central protein in the Cp pathway, and that SNECs from CRSwNP have dysregulated intracellular stores of C3/C3a that can be rapidly mobilized upon stimulation with exogenous irritates. In these proposed studies we will test the hypothesis that dysregulated intracellular Cp signaling in SNEC plays a role in the epithelial cell dysfunction seen in CRSwNP. We will test our hypothesis by executing two inter-related but independent specific aims. In Aim 1 we will determine the role of local and intracellular Cp production in the regulation of human sinonasal epithelial cell inflammation. Sub-Aim 1.1 we will to determine the role of intracellular versus extracellular C3 signaling on SNEC functions. Sub-Aim 1.2 will test the hypothesis that inhibition of Cp-mediated signaling will reduce the pro-inflammatory and hyper-responsive nature of CRSwNP SNEC such that they will function similarly to control subject-derived SNEC. In Aim 2, determine the role local C3 production in the regulation of inflammation and disease severity in a murine model of atopic CRS. Sub-Aim 2.1 will determine the role of locally produced C3 in the sinonasal cavity using our C3-TdT tomato reporter mouse (C3-TdT), and by using a murine model of allergic fungal CRS (Af-CRS) test the key cell types that generate Cp at baseline and under disease conditions. To mimic the human Cp inhibitory therapies utilized in Aim 1, systemic and local C3 will be inhibited with Crry-Ig, and intracellular C3 will be inhibited through the use of intracellular delivery of Crry-Ig with a novel cell penetrating carrier protein (Feldan Shuttle). Using these novel approaches, we will dissect the impact of epithelial and systemic Cp activity on disease development (sub-Aim 2.2), and the therapeutic potential of inhibiting C3 to reverse established disease (sub-Aim 2.3) using the Af-CRS model. Together these studies will provide mechanistic insights into how elevated epithelial intracellular and locally produced Cp can shape the local immune microenvironment.